Termites are xylophages, being able to digest a wide variety of lignocellulosic biomass including wood with high lignin content. This ability to feed on recalcitrant plant material is the result of complex symbiotic relationships, which involve termite-specific gut microbiomes. Therefore, these represent a potential source of microorganisms for the bioconversion of lignocellulose in bioprocesses targeting the production of carboxylates. In this study, gut microbiomes of four termite species were studied for their capacity to degrade wheat straw and produce carboxylates in controlled bioreactors. All of the gut microbiomes successfully degraded lignocellulose and up to 45% w/w of wheat straw degradation was observed, with the Nasutitermes ephratae gut-microbiome displaying the highest levels of wheat straw degradation, carboxylate production and enzymatic activity. Comparing the 16S rRNA gene diversity of the initial gut inocula to the bacterial communities in lignocellulose degradation bioreactors revealed important changes in community diversity. In particular, taxa such as Spirochaetes and Fibrobacteres that were highly abundant in the initial gut inocula were replaced by Firmicutes and Proteobacteria at the end of incubation in wheat straw bioreactors. Overall, this study demonstrates that termite-gut microbiomes constitute a reservoir of lignocellulose-degrading bacteria that can be harnessed in artificial conditions for biomass conversion processes that lead to the production of useful molecules.

The overall kinetics of retting, a spontaneous fermentation of cassava roots performed in central Africa, was investigated in terms of microbial-population evolution and biochemical and physicochemical parameters. During the traditional process, endogenous cyanogens were almost totally degraded, plant cell walls were lysed by the simultaneous action of pectin methylesterase and pectate lyase, and organic acids (C 2 to C 4) were produced. Most microorganisms identified were found to be facultative anaerobes which used the sugars (sucrose, glucose, and fructose) present in the roots as carbon sources. After 24 h of retting, the fermentation reached an equilibrium that was reproducible in all the spontaneous fermentations studied. Lactic acid bacteria were largely predominant (over 99% of the total flora after 48 h) and governed the fermentation. The epiphytic flora was first replaced by Lactococcus lactis, then by Leuconostoc mesenteroides, and finally, at the end of the process, by Lactobacillus plantarum. These organisms produced ethanol and high concentrations of lactate, which strongly acidified the retting juice. In addition, the rapid decrease in partial oxygen pressure rendered the process anaerobic. Strict anaerobes, such as Clostridium spp., developed and produced the volatile fatty acids (mainly butyrate) responsible, together with lactate, for the typical flavor of retted cassava. Yeasts (mostly Candida spp.) did not seem to play a significant role in the process, but their increasing numbers in the last stage of the process might influence the flavor and the preservation of the end products. MATERIALS AND METHODS Origin of the plant material. Cassava roots (Manihot esculenta var. MM86) were harvested near Brazzaville, Congo, 15 months after planting. Retting procedures. Approximatively 100 kg of roots was washed, peeled, and placed in a 200-liter barrel filled with rainwater, which was left at ambient temperature. This retting procedure was repeated four times, always yielding similar results; the data presented are from a single representative retting. Sample preparation for bacterial enumeration. Sampling was performed every 12 h for the first 2 days and then daily until retting was completed. Six randomly selected root sections were cut into 0.5-cm-diameter cubes and mixed under sterile conditions. A 60-g sample was diluted in 540 ml of sterile peptonized water reduced by boiling and addition of cysteine-HCl (0.1% [wt/vol]); this corresponded to a 10 Ϫ1 dilution. The solution was then mixed in a blender (Turnmix ME 88; SOFRACA,

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Prompted by our limited understanding of the degradation of lignin and lignin-derived aromatic metabolites in termites, we studied the metabolism of monoaromatic model compounds by termites and their gut microflora. Feeding trials performed with [ring-U-14 C]benzoic acid and [ring-U-14 C]cinnamic acid revealed the general ability of termites of the major feeding guilds (wood and soil feeders and fungus cultivators) to mineralize the aromatic nucleus. Up to 70% of the radioactive label was released as 14 CO 2 ; the remainder was more or less equally distributed among termite bodies, gut contents, and feces. Gut homogenates of the wood-feeding termites Nasutitermes lujae (Wasmann) and Reticulitermes flavipes (Kollar) mineralized ringlabeled benzoic or cinnamic acid only if oxygen was present. In the absence of oxygen, benzoate was not attacked, and cinnamate was only reduced to phenylpropionate. Similar results were obtained with other, nonlabeled lignin-related phenylpropanoids (ferulic, 3,4-dihydroxycinnamic, and 4-hydroxycinnamic acids), whose ring moieties underwent degradation only if oxygen was present. Under anoxic conditions, the substrates were merely modified (by side chain reduction and demethylation), and this modification occurred at the same time as a net accumulation of phenylpropanoids formed endogenously in the gut homogenate, a phenomenon not observed under oxic conditions. Enumeration by the most-probable-number technique revealed that each N. lujae gut contained about 10 5 bacteria that were capable of completely mineralizing aromatic substrates in the presence of oxygen (about 10 8 bacteria per ml). In the absence of oxygen, small numbers of ring-modifying microorganisms were found (<50 bacteria per gut), but none of these microorganisms were capable of ring cleavage. Similar results were obtained with gut homogenates of R. flavipes, except that a larger number of anaerobic ring-modifying microorganisms was present (>5 ؋ 10 3 bacteria per gut). Neither inclusion of potential cosubstrates (H 2 , pyruvate, lactate) nor inclusion of hydrogenotrophic partner organisms resulted in anoxic ring cleavage in most-probable-number tubes prepared with gut homogenates of either termite. The oxygen dependence of aromatic ring cleavage by the termite gut microbiota is consistent with the presence, and uptake by microbes, of O 2 in the peripheral region of otherwise anoxic gut lumina (as reported in the accompanying paper [A. Brune, D. Emerson, and J. A. Breznak, Appl. Environ. Microbiol. 61:2681-2687, 1995]). Taken together, our results indicate that microbial degradation of plant aromatic compounds can occur in termite guts and may contribute to the carbon and energy requirement of the host.

In the tropics, termites are major players in the mineralization of organic matter leading to the production of greenhouse gases including nitrous oxide (N2O). Termites have a wide trophic diversity and their N-metabolism depends on the feeding guild. This study assessed the extent to which N2O emission levels were determined by termite feeding guild and tested the hypothesis that termite species feeding on a diet rich in N emit higher levels of N2O than those feeding on a diet low in N. An in-vitro incubation approach was used to determine the levels of N2O production in 14 termite species belonging to different feeding guilds, collected from a wide range of biomes. Fungus-growing and soil-feeding termites emit N2O. The N2O production levels varied considerably, ranging from 13.14 to 117.62 ng N2O-N d-1 (g dry wt.)-1 for soil-feeding species, with Cubitermes spp. having the highest production levels, and from 39.61 to 65.61 ng N2O-N d-1 (g dry wt.)-1 for fungus-growing species. Wood-feeding termites were net N2O consumers rather than N2O producers with a consumption ranging from 16.09 to 45.22 ng N2O-N d-1 (g dry wt.)-1. Incubating live termites together with their mound increased the levels of N2O production by between 6 and 13 fold for soil-feeders, with the highest increase in Capritermes capricornis, and between 14 and 34 fold for fungus-growers, with the highest increase in Macrotermes muelleri. Ammonia-oxidizing (amoA-AOB and amoA-AOA) and denitrifying (nirK, nirS, nosZ) gene markers were detected in the guts of all termite species studied. No correlation was found between the abundance of these marker genes and the levels of N2O production from different feeding guilds. Overall, these results support the hypothesis that N2O production rates were higher in termites feeding on substrates with higher N content, such as soil and fungi, compared to those feeding on N-poor wood.